A group of researchers identified a series of genetic mechanisms for acquired resistance to a new class of agents, non-covalent Bruton’s tyrosine kinase (BTK) inhibitors. In particular, resistance to non-covalent BTK inhibitors arose through on-target BTK mutations and downstream PLCγ2 mutations that allowed escape from BTK inhibition. A proportion of these mutations also conferred resistance across clinically approved covalent BTK inhibitors. More analyses of larger samples are necessary to characterise the frequency of these genetic events and how they will influence treatment options according to Dr. Justin Taylor of the Sylvester Comprehensive Cancer Center in Miami, FL, US and Dr. Omar Abdel-Wahab of the Memorial Sloan Kettering Cancer Center in New York, NY, US and colleagues who published the findings in the 24th February 2022 issue of The New England Journal of Medicine.

Covalent, irreversible BTK inhibitors bind to the C481 residue of BTK and block the ATP-binding pocket, thereby preventing catalytic activity. Despite excellent outcomes for patients with chronic lymphocytic leukaemia (CLL) treated with covalent BTK inhibitors, resistance is ultimately acquired in many patients. Resistance to covalent BTK inhibitors is best understood in CLL, in which mutations at the BTK C481 amino acid residue impair drug binding and thereby restore the catalytic activity of BTK. In addition, activating mutations in PLCγ2, a direct substrate of BTK, render malignant cells less reliant on BTK.

Non-covalent, reversible BTK inhibitors were developed to improve on the pharmacologic properties of covalent BTK inhibitors while also maintaining potency against BTK C481 mutations. These agents do not require binding to the BTK C481 residue and effectively inhibit both wild-type and mutant BTK with C481 substitutions. The phase I/II study of pirtobrutinib showed promising efficacy for patients with B-cell malignancy who had previously been treated with covalent BTK inhibitors with 62% response in patients with CLL, including patients with or without BTK C481 mutations.

Mechanisms of resistance to non-covalent BTK inhibitors are currently not well understood. It prompted the researchers to identify the genetic mechanisms and transcriptional characteristics of CLL in patients with clinical resistance to non-covalent BTK inhibitors.

The study team performed genomic analyses of pretreatment specimens, as well as specimens obtained at the time of disease progression from patients with CLL who had been treated with the non-covalent BTK inhibitor pirtobrutinib. Structural modelling, BTK-binding assays, and cell-based assays were conducted to study mutations that confer resistance to non-covalent BTK inhibitors.

Among 55 treated patients, the study team identified 9 patients with relapsed or refractory CLL and acquired mechanisms of genetic resistance to pirtobrutinib. Mutations (V416L, A428D, M437R, T474I, and L528W) were clustered in the kinase domain of BTK and that conferred resistance to both non-covalent BTK inhibitors and certain covalent BTK inhibitors. Mutations in BTK or PLCγ2, a signalling molecule and downstream substrate of BTK, were found in all 9 patients. Transcriptional activation reflecting B-cell–receptor signalling persisted despite continued therapy with non-covalent BTK inhibitors.

The authors wrote that they identified a cluster of mutations in BTK outside the C481 residue, as well as mutations in PLCγ2, that confer resistance to non-covalent BTK inhibitors in patients with CLL. Although initially found in patients with acquired resistance to pirtobrutinib, the series of mutations described in the report confer resistance to a wide array of non-covalent BTK inhibitors in clinical development, including ARQ-531, fenebrutinib, and vecabrutinib, as well as to many existing covalent BTK inhibitors.

It is important to note that the resistance is assessed in only 9 patients with relapsed CLL in an ongoing study that has enrolled more than 250 patients with CLL. Most patients in the overall study have not yet had a relapse, and therefore the present study has analyzed only the patients who have had the earliest relapses. It is unclear whether similar mechanisms of resistance will be observed in patients who have had much longer disease control. Given that the mutations occurred in heavily pretreated patients, it will be critical to evaluate whether similar resistance mechanisms arise in patients receiving non-covalent BTK inhibitors without any previous treatment with covalent BTK inhibitors in earlier lines of therapy.

Covalent BTK inhibitors have transformed the treatment of multiple B-cell malignancies, especially CLL. However, resistance can arise through multiple mechanisms, including acquired mutations in BTK at residue C481, the binding site of covalent BTK inhibitors. Non-covalent BTK inhibitors overcome this mechanism and other sources of resistance. Overall, the development of non-covalent BTK inhibitors represents a promising therapeutic advance for patients with CLL and other B-cell malignancies that have previously been treated with covalent BTK inhibitors.

The study was supported by the American Society of Hematology, the Robert Wood Johnson Foundation, the Doris Duke Charitable Foundation, the Edward P. Evans Foundation, the Society of MSK, grants from the US National Cancer Institute, the Lymphoma Foundation, Fondation de France, the Lymphoma Research Foundation, the CLL Society, and Loxo Oncology.


Wang E, Mi X, Thompson MC, et al. Mechanisms of Resistance to Noncovalent Bruton’s Tyrosine Kinase Inhibitors. N Engl J Med 2022;386:735-743.